Measurement of nitroglycerine-induced vasodilation has been performed to differentiate endothelium-dependent vasodilation from endothelium-independent vasodilation as a control test for flow-mediated vasodilation (FMD). Recently, nitroglycerine-induced vasodilation per se has been reported to be a useful marker of the grade of atherosclerosis. The present study aimed to evaluate the prognostic value of FMD combined with nitroglycerine-induced vasodilation for future cardiovascular events. We measured FMD and nitroglycerine-induced vasodilation in 402 subjects, including patients with cardiovascular diseases. During a median follow-up period of 32.3 months, 38 first major cardiovascular events (death from cardiovascular causes, acute myocardial infarction, stroke, and coronary revascularization) occurred. Receiver-operator characteristic curve analysis revealed that FMD alone and nitroglycerine-induced vasodilation alone can predict cardiovascular events with areas under the curve of 0.671 (cutoff 3.3%) and 0.692 (cutoff 11.6%), respectively. FMD combined with nitroglycerine-induced vasodilation predicts cardiovascular events with an area under the curve of 0.701. After adjustment for age, sex, and cardiovascular risk factors, above cutoff FMD (≥3.3%) and below cutoff nitroglycerine-induced vasodilation (<11.6%; hazard ratio, 5.55; 95% confidence interval, 1.61–25.46; P=0.006) and below cutoff FMD (<3.3%) and below cutoff nitroglycerine-induced vasodilation (<11.6%; hazard ratio, 7.20; 95% confidence interval, 2.37–31.36; P<0.001) remained strong independent indicator of cardiovascular events. These findings suggest that the combination of FMD and nitroglycerine-induced vasodilation measurements can more accurately predict cardiovascular events compared with FMD alone.
Endothelial dysfunction is the initial step of atherosclerosis and plays an important role in the development of this condition. Measurement of flow-mediated vasodilation (FMD) of the brachial artery is a useful method for assessing endothelial function in humans. Appropriate interventions, including pharmacological therapy, supplementation therapy, and lifestyle modifications, are effective for improving endothelial function assessed by FMD in patients with cardiovascular diseases. In addition, several lines of evidence have shown that FMD is an independent predictor of future cardiovascular events.
Nitroglycerine-induced vasodilation, an index of endothelium-independent vasodilation, assessed by sublingual administration of nitroglycerine has been used as a control test for FMD measurement to differentiate endothelium-dependent vasodilation from endothelium-independent vasodilation because both endogenous nitric oxide and administered nitroglycerine act on vascular smooth muscle cells. Recently, we demonstrated that nitroglycerine-induced vasodilation of the brachial artery decreased in relation to cumulative cardiovascular risk factors and was significantly correlated with cardiovascular risk factors as well as FMD in general population, including healthy subjects and patients with cardiovascular disease. In addition, Schächinger et al showed that impaired nitroglycerine-induced vasodilation of coronary arteries is associated with increased risk of cardiovascular events. These findings suggest that nitroglycerine-induced vasodilation may be not only a control test for FMD measurement but also a useful marker of the grade of atherosclerosis.
Although it is well known that FMD is an independent predictor of future cardiovascular events, some studies have shown that FMD is not associated with future cardiovascular events in cases of a relatively low grade of atherosclerosis and in cases of a severe stage of atherosclerosis after adjusting for various confounders. The reasons for the discrepant results remain unclear.
It is expected that nitroglycerine-induced vasodilation would predict future cardiovascular events. The combination of measurements of FMD and nitroglycerine-induced vasodilation would be a more useful biomarker reflecting vascular function and would improve the predictive value of future cardiovascular events compared with measurement of FMD alone. In this study, we evaluated the prognostic values of measurements of FMD alone, nitroglycerine-induced vasodilation alone, and FMD combined with nitroglycerine-induced vasodilation for future cardiovascular events.
Between July 2007 and October 2013, a total of 402 subjects were enrolled from people who underwent health-screening examinations at Hiroshima University Hospital. All subjects have an obligation to undergo health screening every year under regulation of the society-managed health insurance union in Japan. Hypertension was defined as systolic blood pressure of >140 mm Hg and diastolic blood pressure of >90 mm Hg, in a sitting position, on at least 3 different occasions. Normotension was defined as systolic blood pressure of <140 mm Hg and diastolic blood pressure of <90 mm Hg. Diabetes mellitus was defined according to the American Diabetes Association or a previous diagnosis of diabetes mellitus. Dyslipidemia was defined according to the third report of the National Cholesterol Education Program. Framingham risk score was calculated by the following points of risk factors: age, total cholesterol level, high-density lipoprotein cholesterol level, systolic blood pressure, and smoking status. The ethical committees of our institutions approved the study protocol. Written informed consent for participation in the study was obtained from all subjects.
We assessed vascular function by measurements of FMD and nitroglycerine-induced vasodilation in all subjects. Subjects were instructed to abstain from eating, alcohol, smoking, and caffeine for at least 12 hours before the measurements. The subjects were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature of 22°C–25°C) throughout the study. A 23-gauge polyethylene catheter was inserted into the left deep antecubital vein to obtain blood samples. Thirty minutes after resting in the supine position, FMD and nitroglycerine-induced vasodilation were measured.
From December 2013 to May 2014, we collected the information on potential outcomes or adverse events from medical records and from 1 telephone survey. We obtained complete follow-up data on 402 subjects. We first assessed the associations of FMD alone, nitroglycerine-induced vasodilation alone, and FMD combined with nitroglycerine-induced vasodilation and first major cardiovascular events (death from cardiovascular causes, acute myocardial infarction, stroke, and coronary revascularization) and then assessed the associations with death from cardiovascular causes, acute myocardial infarction, stroke, coronary revascularization, and hospitalization for heart failure.
A high-resolution ultrasonography equipment (UNEXEF18G; UNEX Co, Nagoya, Japan) was used to evaluate FMD and nitroglycerine-induced vasodilation. Additional details are available in the online-only Data Supplement.
Because there is no previous study investigating the association between FMD combined with nitroglycerine-induced vasodilation and cardiovascular events, we enrolled all subjects in whom vascular function was assessed. Some variables in the data set had missing data: body mass index (BMI, n=2), serum concentrations of total cholesterol (n=10), triglycerides (n=11), high-density lipoprotein cholesterol (n=10), low-density lipoprotein cholesterol (n=10), glucose (n=50), and Framingham risk score (n=10). Missing data were not imputed and all continuous variables were analyzed using observed-case analysis. Results are presented as means±SD for continuous variables and as percentages for categorical variables. Statistical significance was set at a level of P<0.05. Continuous variables were compared by using ANOVA for multiple groups. Categorical variables were compared by means of the χ 2 test. The receiver-operator characteristic curve analyses were performed to assess the sensitivity and specificity of measurement of FMD alone, nitroglycerine-induced vasodilation alone, and FMD combined with nitroglycerine-induced vasodilation for predicting first major cardiovascular events. To take time-to-event into consideration, we calculated the Harrell C index. Time-to-event end-point analyses were performed by using the Kaplan–Meier method. We categorized subjects into 4 groups according to the cutoff value of FMD and the cutoff value of nitroglycerine-induced vasodilation. Cutoff values were derived from the receiver-operator characteristic curves. A log-rank test was used to compare survival in the groups. A post hoc power analysis was performed to assess the adequacy of sample size using the observed cohort size. As the primary analysis, we evaluated the associations between FMD combined with nitroglycerine-induced vasodilation and first major cardiovascular events after adjustment for age, sex, and cardiovascular risk factors by using Cox proportional hazard regression analysis. Multiplicity was controlled with a step-down closed testing procedure for the first major cardiovascular events with the following comparisons: group 1 versus group 4, group 1 versus group 2, group 1 versus group 3. As the sensitivity analysis, we performed exploratory analysis to evaluate the prognostic value of FMD combined with nitroglycerine-induced vasodilation before and after adjustment for age and sex. In a second sensitivity analysis, the proportional hazards assumption was confirmed by inspection of Schoenfeld residuals and log-log plotting. In a third sensitivity analysis, the increased discriminative value of FMD combined with nitroglycerine-induced vasodilation was further examined by using the net reclassification improvement (NRI). Subjects were initially stratified as low-risk, intermediate-risk, and high-risk groups according to the Framingham risk score: low risk (0 to <6%), intermediate risk (6% to 20%), and high risk (>20%). Subjects could then be reclassified into different categories with the addition of data for FMD combined with nitroglycerine-induced vasodilation. We assessed the number of subjects reclassified and also calculated NRI. The data were processed using the software package Stata, version 9 (Stata Co, College Station, TX).
The baseline characteristics of the 402 subjects are summarized in Table 1. Of the 402 subjects, 248 (61.7%) were men and 154 (38.3%) were women. Two hundred and fifty-six (63.7%) had hypertension, 237 (59.0%) had dyslipidemia, 127 (31.6%) had diabetes mellitus, and 103 (26.4%) were current smokers. The mean values of FMD and nitroglycerine-induced vasodilation were 4.3±2.7% and 12.5±5.7%, respectively.
